Alcohol abuse and alcoholism are significant public health issues faced by many countries. In the United States alone, alcohol use disorders affect about 14 million people, costing approximately $184 billion a year due to lost wages, legal costs, and medical costs resulting from associated injuries and liver, cardiac, neoplastic, or infectious diseases. Treatment generally consists of pharmacological interventions to manage detoxification, and psychosocial therapies aimed at rehabilitation and reducing alcohol-associated problems. While some treatments are effective in reducing alcohol consumption, it is estimated that 40-70% of patients return to excessive drinking within a year after treatment (Addiction (1996) 91:1773-1796). Advances in neuroscience have excited interest in developing pharmaceuticals to dampen craving for alcohol and provide more effective treatment. So far, only three drugs have been approved for treatment in the United States: Disulfuram, naltrexone and acamprosate. Disulfuram is an irreversible inhibitor of aldehyde dehydrogenase and reinforces a patient's desire to avoid alcohol because when alcohol is consumed, levels of acetaldehyde increase, resulting in nausea, hypotension and flushing. Disulfuram is useful for the short-term treatment of highly motivated and compliant patients, but there is no evidence that it is effective in long-term therapy and it carries a risk of significant liver, cardiac, and nervous system toxicity if taken with alcohol. Naltrexone is a non-selective opioid receptor antagonist that decreases the euphoric and reinforcing effects of alcohol and reduces relapse to drinking, especially when combined with psychotherapy. Although most studies have shown it to be beneficial (Addiction (2001) 96:1565-1573 and references therein), others have not (N Eng J Med (2001) 345:1734-1739 and references therein). Its clinical usefulness appears limited as naltrexone can cause fatigue, sedation, nausea, and abdominal pain in a significant number of patients, its beneficial effects tend to diminish over time, and compliance is variable. Acamprosate can reduce the frequency of drinking and is indicated for maintenance of abstinence (Am J Health Syst Pharm (2004) 61:2272-2279 and references therein). Its mechanism of action is not well understood, though it may mainly act to inhibit NMDA and mGluR5 receptors. Although generally well tolerated, it can cause diarrhea in up to 20% of patients, and it also can cause rash, dizziness, and loss of libido. For both naltrexone and acamprosate compliance is generally low, with only about half of patients completing treatment with either drug. Clearly, there is a need to develop more effective compounds, and several new ones are currently being studied, including the 5HT3 receptor antagonist ondansetron and the anticonvulsant topiramate (Am J Health Syst Pharm (2004) 61:2380-2388 and refs therein).
A large body of preclinical data has accumulated through studies of mice deficient in PKCε and animals treated with a peptide inhibitor of PKCε, supporting therapeutic use of PKCε inhibitors to treat alcohol abuse and alcoholism (Hodge et al., Nat. Neurosci., 1999, 2, 997-1002). Using PKCε null mice, it has been shown that PKCε regulates alcohol consumption, dependence and reward. Restoration of neuronal PKCε by means of tetracycline-regulated transgenic expression elevates alcohol intake to levels observed in wild type mice, indicating that reduced alcohol consumption in PKCε null mice is due to loss of PKCε in adult neurons (Choi et al., J. Neurosci., 2002, 22, 9905-9911). PKCε null mice also show reduced operant self-administration of alcohol and reduced relapse drinking following alcohol deprivation (Olive et al., Eur. J. Neurosci., 2000, 12, 4131-4140). These findings are associated with less severe alcohol withdrawal seizures (Olive et al., Neuroscience, 2001, 103, 171-179) and markedly reduced dopamine release in the nucleus accumbens following ethanol injection (Olive et al., Eur. J. Neurosci., 2000, 12, 4131-4140). These results suggest that the positive rewarding and reinforcing properties of alcohol, as well as the negative effects of alcohol withdrawal could be reduced by PKCε inhibition.
Other studies provide a strong case for therapeutic use of PKCε inhibitors to reduce pain associated with alcoholic polyneuropathy and inflammatory and neuropathic pain. Another study (Dina et al., J. Neurosci., 2000, 20, 8614-8619) analyzed the role of PKCε in alcohol-induced pain using a rat model of alcohol-induced hyperalgesia and found that the hyperalgesia was acutely attenuated by non-selective PKC inhibitors and by a selective peptide inhibitor of PKCε, εV1-2, injected intradermally at the site of nociceptive testing. Another study (Aley et al., J. Neurosci., 2000, 20, 4680-4685) identified a PKCε-mediated mechanism for chronic inflammatory pain. In this case, a nonselective inhibitor of several PKC isozymes and a selective PKCε inhibitor antagonized the prolonged hyperalgesic response in the carrageenan injected rat hindpaw equally.
In addition, evidence indicates that PKCε inhibitors could be useful for the treatment of anxiety, which is commonly associated with alcoholism and may contribute to excessive drinking. Other studies (Hodge et al., Nat. Neurosci., 1999, 2, 997-1002; Hodge et al., J. Clin. Invest., 2002, 110, 1003-1010) examined GABAA receptor function in PKCε null mice and found that these mice are supersensitive to the acute hypnotic effects of barbiturates, benzodiazepines, ethanol and neurosteroids.
A large amount of evidence from preclinical studies and patient tumor analysis has indicated that PKCε is a transforming oncogene and played a critical role in tumor cell proliferation, motility, invasion and drug resistance (Gorin & Pan, Molecular Cancer, 2009, 8, 9). In vitro, overexpression of PKCε has been demonstrated to increase proliferation, motility, and invasion of fibroblasts or immortalized epithelial cells. In addition, xenograft and transgenic animal models have clearly shown that overexpression of PKCε is tumorigenic resulting in metastatic disease. PKCε has been found to be overexpressed in tumor-derived cell lines and histopathological tumor specimens from various organ sites. Activation of PKCε causes up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance (Bourguignon et al., J. Biol. Chem., 2009, 284, 26533-26546). Thus, PKCε inhibitors could be therapeutically useful in treating a variety of cancer including breast, head and neck, prostate and lung cancer either used alone or in combination with standard cancer therapy.
In another study of type 2 diabetes (Schmitz-Peiffer et al., Cell Metabolism, 2007, 6, 320-328), a role for PKCε in 13 cell dysfunction was established. Deletion of PKCεaugmented insulin secretion and prevented glucose intolerance in fat-fed mice. Importantly, a PKCε-inhibitory peptide improved insulin availability and glucose tolerance in diabetic mice. In another study (Samuel et al., J. Clin. Invest., 2007, 117, 739-745), PKCε, but not other isoforms of PKC, was activated in the high-fat fed rats resulting in hepatic steatosis and hepatic insulin resistance. Knocking down PKCε expression by treating the rats with an antisense oligonucleotide against PKCε protected rats from fat-induced hepatic insulin resistance. Therefore, PKCε inhibitors of this invention could be beneficial treating type 2 or type 1 diabete in humans.
In another study (Kaiser et al., Arch. Biochem. Biophys., 2009, 482, 104-11), PKCε was shown to contribute to hepatic steatosis in experimental ethanol-induced and non-alcoholic fatty liver disease using PKCε knockout mice and in wild-type nice that received an anti-sense oligonucleotide to knockdown PKCε expression. The data suggest that activation of PKCε exacerbates hepatic lipid accumulation by inducing insulin resistance. Thus, inhibition of PKCε with the inhibitors of this invention could prevent and/or treat liver diseases.
It was also demonstrated that PKCε is a target to control inflammation and immune-mediated disorders (Aksoy et al., Intern. J. Biochem. Cell Biol., 2004, 36, 183-188). Controlling the kinase activity of PKCε might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin. Furthermore, pharmacological inhibition of PKCε suppressed chronic inflammation in murine cardiac transplantation model (Koyanagi et al., J. Mol. Cell. Cardio., 2007, 43, 517-522). It is possible that the PKCε inhibitors of this invention could be used therapeutically for prevention and treatment of various inflammatory conditions associated with e.g. microbial infection and organ transplantation.
Above mentioned studies provide a strong case for development of PKCε inhibitors to treat diseases or disorders or conditions mentioned above. Given the large number of people who may be helped by such a drug, and given the high cost of these diseases, the benefits to society could be enormous if an effective PKCε inhibitor can be developed.
PKC is a family of serine-threonine kinases with important roles in cell growth, differentiation, ion channel and receptor regulation, gene expression, tumor promotion and apoptosis. The mammalian PKC family contains at least 9 genes grouped into 3 classes: conventional (α, β, γ), novel (δ, ε, η/L, θ), and atypical (ξ, ι/λ) PKC isozymes. PKC inhibitors have been considered attractive as therapeutic agents. A relatively selective inhibitor of PKCβ, LY333531, has undergone a clinical study for the treatment of diabetic retinopathy. The use of PKC inhibitors has been suggested for clinical applications ranging from the treatment of psoriasis to cancer (Expert Opin Investig Drugs (2001) 10:2117-2140 and refs therein). Knowledge of specific roles attributable to individual PKC isozymes has been hampered by the lack of isozyme-selective drugs—there are currently no selective small organic molecule inhibitors of PKCε and the search for selective drugs has been difficult because of several methodological limitations (Biochemical Journal (2003) 371:199-204 and refs therein).
Thus, there is still a strong need for compounds that act as epsilon isozyme-selective inhibitors of the protein PKC.